The Type I IFN system serves as a key component of the host innate antiviral immune response. A major outcome of IFN signaling following viral infection is the induction of hundreds of interferon-stimulated genes (ISGs). Only a handful of ISGs have been characterized with respect to their antiviral effector functions. The importance of IFN effectors to antiviral immunity is underscored by the observation that many viruses subvert host defenses by interfering with ISG induction or function. Therapeutically, IFN is utilized to limit viral replication, but treatment is often ineffective and highly toxic. The negative attributes of IFN therapy, combined with a pressing need for countermeasures against highly pathogenic viruses (many of which have bioterrorism potential), warrant the exploration of cellular antiviral effectors to determine their mechanism of action and spectrum of activity. The primary goals of this project are to identify and characterize antiviral ISGs, with an emphasis on the discovery of broad-spectrum effectors that limit the replication of several category A-C priority viruses. The experimental approach utilizes a lentivirus-based overexpression screen to identify antiviral IFN effectors, focusing initially on alphaviruses (Chikungunya and Sindbis) and flaviviruses (dengue and yellow fever). Fluorescent-based reporter viruses will be used to assess the antiviral activity of ISGs, and viral inhibition will be quantified in a high-throughput manner by flow cytometry or automated fluorescence microscopy. In collaborative efforts with other biodefense investigators, positive hits will be evaluated for their antiviral potential against more highly pathogenic viruses. With a preference for those exhibiting broad-spectrum antiviral activity, detailed mechanism-of-action studies will be conducted. In the long term, small molecule screens will be employed to identify compounds that selectively upregulate expression or potentiate the action of specific antiviral ISGs.